Novel treatment of prostate carcinoma

ABSTRACT

Disclosed herein are naphthoquinone analogs, such as plumbagin, pharmaceutical compositions that include naphthoquinone analogs, such as plumbagin, and methods of treating diseases and/or conditions such as cancer with naphthoquinone analogs, such as plumbagin. Also included are combination therapies wherein a naphthoquinone analog, such as plumbagin, and a hormone therapy agent are provided to a subject suffering from a condition such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 13/813,394filed Mar. 27, 2013, which is a national phase application ofInternational Patent Application No. PCT/US11/46474, filed Aug. 3, 2011,which designated the United States and was written in English and, whichclaims the benefit of priority to U.S. Provisional Application No.61/370,534, filed Aug. 4, 2010. The disclosures of the aforementionedapplications are hereby expressly incorporated by reference in theirentireties.

FIELD OF THE INVENTION

Aspects of the present application relate to the fields of chemistry,biochemistry and medicine. More particularly, disclosed herein arenaphthoquinone analogs, such as plumbagin, pharmaceutical compositionsthat include naphthoquinone analogs, such as plumbagin, and methods oftreating diseases and/or conditions with naphthoquinone analogs, such asplumbagin. Also included are combination therapies, wherein anaphthoquinone analog, such as plumbagin, and a hormone therapy agent,such as a hormonal ablation compound, are provided to a subject having acancer, such as a prostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer develops in the prostate and is typically slow growing;however, some prostate cancers are aggressive. Prostate cancer cells aretypically androgen/testosterone/DHT dependent and may metastasize fromthe prostate to other parts of the body, particularly the bones andlymph nodes. Treatment options for prostate cancer that remains withinthe prostate include watchful waiting/active surveillance, external beamradiation therapy, brachytherapy, cryosurgery, HIFU, and surgery.Hormonal therapy and chemotherapy are often reserved for disease thathas spread beyond the prostate. However, there are exceptions in thatradiation therapy may be used for some advanced tumors, and hormonaltherapy may be used for some early stage tumors.

After one to three years of hormonal therapy, it is common that prostatecancer cells resume growth despite the androgen/testosterone/DHTblockade. Previously referred to as “hormone-refractory prostate cancer”or “androgen-independent prostate cancer,” the term castration-resistantprostate cancer (CRPC) is now commonly used. Chemotherapeutic agents andimmunotherapy have been shown to prolong survival after CRPC but thesurvival benefit is limited. Despite the efforts of many, the need formore cancer treatments, in particular prostate cancer treatments, ismanifest.

SUMMARY

Some embodiments disclosed herein relate to a method of inhibiting ordelaying the growth of prostate cancer by providing a subject havingprostate cancer with a therapeutically effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt of Formula (I), whilereducing the amount of an androgen in the subject. In some embodiments,the amount of androgen can be reduced by providing the subject with ananti-androgen compound, an estrogen, a luteinizing hormone-releasinghormone (LHRH) agonist, or a LHRH antagonist. In some embodiments, theamount of androgen can be reduced by providing the subject with asteroidal anti-androgen or a non-steroidal anti-androgen. In someembodiments, the amount of androgen can be reduced by providing thesubject with cyproterone acetate, abiraterone, finasteride, flutamide,nilutamide, bicalutamide, ethylstilbestrol (DES), megestrol acetate,fosfestrol, estamustine phosphate, leuprolide, triptorelin, goserelin,histrelin, buserelin, abarelix and/or degarelix. In some embodiments,the method of inhibiting or delaying the growth of prostate cancer canreduce the subject's serum testosterone level to between about 20-50ng/dL. In some embodiments, the method of inhibiting or delaying thegrowth of prostate cancer can reduce the subject's serum testosteronelevel to less than about 50 ng/dL. In some embodiments, the method ofinhibiting or delaying the growth of prostate cancer can reduce thesubject's serum testosterone level to less than about 20 ng/dL.

Some embodiments disclosed herein relate to a method for identifying acompound that inhibits or delays prostate cancer cell growth byproviding a pseudo-orthotopic chamber mouse model, wherein the mousemodel has prostate cancer; reducing the level of an androgen in saidmouse model; providing the mouse model with a compound of Formula (I) ora pharmaceutically acceptable salt or a prodrug thereof; and evaluatingwhether the compound is effective in inhibiting the growth of prostatecancer cells.

Some embodiments disclosed herein relate to a method of inhibiting ordelaying the onset of castration-resistant prostate cancer (CRPC) byclassifying a subject as a member of a population that is at risk fordeveloping CRPC; providing said subject with a therapeutically effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt of Formula (I), while reducing the amount of an androgen in saidsubject; and evaluating an inhibition or delay of prostate cancer cellgrowth or a marker thereof or the onset of CRPC.

Some embodiments disclosed herein relate to a method of identifying acompound that inhibits or delays prostate cancer cell growth bycontacting prostate cancer cells with a compound of Formula (I) in theabsence of androgen; determining the presence or absence of aninhibition or delay in prostate cancer cell growth; and classifying thecompound into a population that inhibits or delays prostate cancer cellgrowth in the absence of androgen, or into a population that does notinhibit or delay prostate cancer cell growth.

Some embodiments disclosed herein relate to a method of making aprostate cancer therapeutic by contacting prostate cancer cells with acompound of Formula (I) in the absence of androgen; determining thepresence or absence of an inhibition or delay in prostate cancer cellgrowth; selecting a compound of Formula (I) that inhibits prostatecancer cell growth in the absence of androgen; and formulating thecompound that inhibits or delays prostate cancer cell growth in theabsence of androgen for administration to a subject suffering fromprostate cancer.

Some embodiments disclosed herein relate to a combination of a compoundof Formula (I) or a pharmaceutically acceptable salt of Formula (I) anda hormone therapy agent for inhibiting or delaying prostate cancer cellgrowth or the onset of castration-resistant prostate cancer (CRPC). Insome embodiments, the hormone therapy agent can be cyproterone acetate,abiraterone, finasteride, flutamide, nilutamide, bicalutamide,ethylstilbestrol (DES), megestrol acetate, fosfestrol, estamustinephosphate, leuprolide, triptorelin, goserelin, histrelin, buserelin,abarelix or degarelix or any combination of one or more of saidcompounds.

Some embodiments disclosed herein relate to a combination of a compoundof Formula (I) or a pharmaceutically acceptable salt of Formula (I) anda hormone therapy agent for use in decreasing prostate tumor size. Insome embodiments, the hormone therapy agent can be cyproterone acetate,abiraterone, finasteride, flutamide, nilutamide, bicalutamide,ethylstilbestrol (DES), megestrol acetate, fosfestrol, estamustinephosphate, leuprolide, triptorelin, goserelin, histrelin, buserelin,abarelix or degarelix or any combination of one or more of saidcompounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of naphthoquinone analogs on PTEN-P2/GFP cellproliferation.

FIG. 2 shows the effect of naphthoquinone analogs on PTEN-P2/GFP cellproliferation.

FIG. 3 shows the dose response of plumbagin in PTEN-P2/GFP cells.

FIG. 4 compares the growth of tumors without treatment, with castrationalone, with plumbagin alone, and the combination of castration andplumbagin.

FIG. 5 shows the effect of plumbagin at 0.1 mg/kg, 0.3 mg/kg and 1mg/kg, given in combination with castration.

FIG. 6 illustrates the effect of adding plumbagin after surgicalcastration.

FIG. 7 illustrates increasing apoptosis (AP) and mitosis (MI) afterdaily administration of plumbagin ip (2 mg/kg).

FIG. 8 illustrates the effect of plumbagin in human LNCaP cells in theabsence of dihydrotestosterone.

DETAILED DESCRIPTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

As used herein, any “R” group(s) such as, without limitation, R, R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ representsubstituents that can be attached to the indicated atom. An R group maybe substituted or unsubstituted.

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in an alkyl, alkenyl or alkynyl group, orthe number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, thealkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of thecycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of theheteroaryl or ring of the heteroalicyclyl can contain from “a” to “b”,inclusive, carbon atoms. Thus, for example, a “C₁ to C₄ alkyl” grouprefers to all alkyl groups having from 1 to 4 carbons, that is, CH₃—,CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and(CH₃)₃C—. If no “a” and “b” are designated with regard to an alkyl oralkenyl group, the broadest range described in these definitions is tobe assumed.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms(whenever it appears herein, a numerical range such as “1 to 20” refersto each integer in the given range; e.g., “1 to 20 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl andhexyl. The alkyl group may be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted.

The term “halogen” as used herein, means any one of the radio-stableatoms of column 7 of the Periodic Table of the Elements, such as,fluorine, chlorine, bromine and iodine.

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted or substituted with one or more of theindicated substituents. Likewise, when a group is described as being“unsubstituted or substituted” if substituted, the substituent may beselected from one or more the indicated substituents. If no substituentsare indicated, it is meant that the indicated “optionally substituted”or “substituted” group may be substituted with one or more group(s)individually and independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio,arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl,haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, amino, mono-substituted amino group anddi-substituted amino group, and protected derivatives thereof.

The term “naphthoquinone analog” refers to a compound of Formula (I)wherein R¹, R², R³, R⁴, R⁵, and R⁶ are as defined herein.

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid andphosphoric acid. Pharmaceutical salts can also be obtained by reacting acompound with an organic acid such as aliphatic or aromatic carboxylicor sulfonic acids, for example formic, acetic, succinic, lactic, malic,tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic,p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceuticalsalts can also be obtained by reacting a compound with a base to form asalt such as an ammonium salt, an alkali metal salt, such as a sodium ora potassium salt, an alkaline earth metal salt, such as a calcium or amagnesium salt, a salt of organic bases such as dicyclohexylamine,N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine,cyclohexylamine, triethanolamine, ethylenediamine, and salts with aminoacids such as arginine and lysine.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be disatereomerically pure, disatereomerically enriched, ormay be stereoisomeric mixtures. In addition it is understood that, inany compound described herein having one or more double bond(s)generating geometrical isomers that can be defined as E or Z, eachdouble bond may independently be E or Z a mixture thereof. Likewise, itis understood that, in any compound described, all tautomeric forms arealso intended to be included.

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Pharmaceutical compositions can also beobtained by reacting compounds with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid and salicylic acid. Pharmaceutical compositionswill generally be tailored to the specific intended route ofadministration.

The term “physiologically acceptable” defines a carrier, diluent orexcipient that does not abrogate the biological activity and propertiesof the compound.

As used herein, a “carrier” refers to a compound that facilitates theincorporation of a compound into cells or tissues. For example, withoutlimitation, dimethyl sulfoxide (DMSO) is a commonly utilized carrierthat facilitates the uptake of many organic compounds into cells ortissues of a subject.

As used herein, a “diluent” refers to an ingredient in a pharmaceuticalcomposition that lacks pharmacological activity but may bepharmaceutically necessary or desirable. For example, a diluent may beused to increase the bulk of a potent drug whose mass is too small formanufacture and/or administration. It may also be a liquid for thedissolution of a drug to be administered by injection, ingestion orinhalation. A common form of diluent in the art is a buffered aqueoussolution such as, without limitation, phosphate buffered saline thatmimics the composition of human blood.

As used herein, an “excipient” refers to an inert substance that isadded to a pharmaceutical composition to provide, without limitation,bulk, consistency, stability, binding ability, lubrication,disintegrating ability etc., to the composition. A “diluent” is a typeof excipient

As used herein, a “subject” refers to an animal that is the object oftreatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles and, in particular, mammals. “Mammal” includes, withoutlimitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats,cows, horses, primates, such as monkeys, chimpanzees, and apes, and, inparticular, humans. In some embodiments, the subject is human.

As used herein, the terms “treating,” “treatment,” “therapeutic,” or“therapy” do not necessarily mean total cure or abolition of the diseaseor condition. Any alleviation of any undesired signs or symptoms of adisease or condition, to any extent can be considered treatment and/ortherapy. Furthermore, treatment may include acts that may worsen thepatient's overall feeling of well-being or appearance.

The term “therapeutically effective amount” is used to indicate anamount of an active compound, or pharmaceutical agent, that elicits thebiological or medicinal response indicated. For example, atherapeutically effective amount of compound can be the amount needed toprevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. This response may occur in atissue, system, animal or human and includes alleviation of the signs orsymptoms of the disease being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, in view of the disclosure provided herein. Thetherapeutically effective amount of the compounds disclosed hereinrequired as a dose will depend on the route of administration, the typeof animal, including human, being treated, and the physicalcharacteristics of the specific animal under consideration. The dose canbe tailored to achieve a desired effect, but will depend on such factorsas weight, diet, concurrent medication and other factors which thoseskilled in the medical arts will recognize.

As used herein, the term “hormone therapy agent” refers toanti-androgens (including steroidal anti-androgens and non-steroidalanti-androgens), estrogens, luteinizing hormone-releasing hormone (LHRH)agonists, and LHRH antagonists, as well as, hormonal ablation therapy.Exemplary hormone therapy agents include, but are not limited to,cyproterone acetate, abiraterone, finasteride, flutamide, nilutamide,bicalutamide, ethyl stilbestrol (DES), megestrol acetate, fosfestrol,estamustine phosphate, leuprolide, triptorelin, goserelin, histrelin,buserelin, abarelix and degarelix.

As used in this specification, whether in a transitional phrase or inthe body of the claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases “having at least” or“including at least.” When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of acompound, composition or device, the term “comprising” means that thecompound, composition or device includes at least the recited featuresor components, but may also include additional features or components.The section below describes some of the compounds that can be used totreat cancer, or inhibit or delay the growth of cancer cells, especiallyprostate cancer cells alone or in combination with one or more androgendeprivation therapies (e.g., castration, hormonal castration, hormonalablation, or hormone therapy).

II. Compounds of Formula (I)

Some embodiments disclosed herein relate to a compound of Formula (I), apharmaceutically acceptable salt thereof, and methods of using thesecompounds with and without a hormone therapy agent, as described herein,to inhibit, delay, treat, or prevent prostate cancer cell growth orprostate cancer in a subject in need thereof. Formula (I):

wherein: R¹ can be selected from hydrogen, halogen, an optionallysubstituted C₁₋₁₈ alkyl, an optionally substituted C₂₋₁₈ alkenyl, —OR⁷and —SR⁸; R² can be selected from hydrogen, halogen, an optionallysubstituted C₁₋₆ alkyl, an optionally substituted C₂₋₆ alkenyl, —OR⁹ and—SR¹⁰; R³ can be selected from hydrogen, an optionally substituted C₁₋₆alkyl, and —OR¹¹; R⁴ can be selected from hydrogen, an optionallysubstituted C₁₋₆ alkyl, and —OR¹²; R⁵ can be selected from hydrogen, anoptionally substituted C₁₋₆ alkyl, and —OR¹³; R⁶ can be selected fromhydrogen, an optionally substituted C₁₋₆ alkyl, and —OR¹⁴; and R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ can be independently selected fromhydrogen and an optionally substituted C₁₋₆ alkyl.

In some embodiments, R¹ can be hydrogen. In some embodiments, R¹ can behalogen. In some embodiments, R¹ can be chloro. In some embodiments, R¹can be an optionally substituted C₁₋₁₈ alkyl. Examples of optionallysubstituted C₁₋₁₈-alkyls include, but are not limited to, optionallysubstituted variants of the following: methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl,nonanyl, decanyl, undecanyl, dodecanyl, tridecanyl, tetradecanyl,pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl, and phytanyl.Optionally substituted C₁₋₁₈-alkyls can be branched or straight-chained.In some embodiments, R¹ can be an optionally substituted C₁₋₆ alkyl. Insome embodiments, R¹ can be methyl. In some embodiments, R¹ can bet-butyl. In some embodiments, R¹ can be an optionally substituted C₂₋₁₈alkenyl. Examples of optionally substituted C₂₋₁₈-alkenyls include, butare not limited to, optionally substituted variants of the following:ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl,pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, and phytenyl.Optionally substituted C₂₋₁₈-alkenyls can be branched orstraight-chained, and can include one or more double bonds. In someembodiments, R¹ can be an optionally substituted C₂₋₆ alkenyl. In someembodiments, R¹ can be —OR⁷, wherein R⁷ is hydrogen. In someembodiments, R¹ can be —OR⁷, wherein R⁷ is an optionally substitutedC₁₋₆ alkyl. In some embodiments, R¹ can be —OR⁷, wherein R⁷ is methyl.In some embodiments, R¹ can be —SR⁸, wherein R⁸ is hydrogen. In someembodiments, R¹ can be —SR⁸, wherein R⁸ is an optionally substitutedC₁₋₆ alkyl. In some embodiments, R¹ can be —SR⁸, wherein R⁸ is C₁₋₆alkyl optionally substituted with hydroxy. In some embodiments, R¹ canbe —SR⁸, wherein R⁸ is —CH₂CH₂OH.

In some embodiments, R² can be hydrogen. In some embodiments, R² can behalogen. In some embodiments, R² can be chloro. In some embodiments, R²can be an optionally substituted C₁₋₆ alkyl. Examples of optionallysubstituted C₁₋₆-alkyls include optionally substituted variants of thefollowing: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, pentyl (branched and straight-chained), and hexyl (branchedand straight-chained). In some embodiments, R² can be methyl. In someembodiments, R² can be an optionally substituted C₂₋₆ alkenyl. Examplesof optionally substituted C₂₋₆-alkenyls include optionally substitutedvariants of the following: ethenyl, propenyl, butenyl, pentenyl(branched and straight-chained), and hexenyl (branched andstraight-chained). In some embodiments, R² can be —CH₂—CH═C(CH₃)₂. Insome embodiments, R² can be —OR⁹, wherein R⁹ is hydrogen. In someembodiments, R² can be —OR⁹, wherein R⁹ is an optionally substitutedC₁₋₆ alkyl. In some embodiments, R² can be —OR⁹, wherein R⁹ is methyl.In some embodiments, R² can be —SR¹⁰, wherein R¹⁰ is hydrogen. In someembodiments, R² can be —SR¹⁰, wherein R¹⁰ is an optionally substitutedC₁₋₆ alkyl. In some embodiments, R² can be —SR¹⁰, wherein R¹⁰ is C₁₋₆alkyl optionally substituted with hydroxy. In some embodiments, R² canbe —SR¹⁰, wherein R¹⁰ is —CH₂CH₂OH.

In some embodiments, R³ can be hydrogen. In some embodiments, R³ can bean optionally substituted C₁₋₆ alkyl. In some embodiments, R³ can be—OR¹¹, wherein R¹¹ is hydrogen. In some embodiments, R³ can be —OR¹¹,wherein R¹¹ is an optionally substituted C₁₋₆ alkyl.

In some embodiments, R⁴ can be hydrogen. In some embodiments, R⁴ can bean optionally substituted C₁₋₆ alkyl. In some embodiments, R⁴ can bet-butyl. In some embodiments, R⁴ can be —OR¹², wherein R¹² is hydrogen.In some embodiments, R⁴ can be —OR¹², wherein R¹² is an optionallysubstituted C₁₋₆ alkyl.

In some embodiments, R⁵ can be hydrogen. In some embodiments, R⁵ can bean optionally substituted C₁₋₆ alkyl. In some embodiments, R⁵ can be—OR¹³, wherein R¹³ is hydrogen. In some embodiments, R⁵ can be —OR¹³,wherein R¹³ is an optionally substituted C₁₋₆ alkyl.

In some embodiments, R⁶ can be hydrogen. In some embodiments, R⁶ can bean optionally substituted C₁₋₆ alkyl. In some embodiments, R⁶ can be—OR¹⁴, wherein R¹⁴ is hydrogen. In some embodiments, R⁶ can be —OR¹⁴,wherein R¹³ is an optionally substituted C₁₋₆ alkyl.

In some embodiments, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ can beindependently selected from hydrogen. In some embodiments, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ can be independently selected from C₁₋₆alkyl. In some embodiments, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ canbe independently selected from C₁₋₆ alkyl, wherein the C₁₋₆ alkyl can beoptionally substituted with a group selected from halogen, hydroxy, andC₁₋₄ alkyl.

In some embodiments, R¹ can be selected from hydrogen, halogen, anoptionally substituted C₁₋₆ alkyl, —OR⁷ and —SR⁸; R² can be selectedfrom hydrogen, halogen, an optionally substituted C₁₋₆ alkyl, —OR⁹ and—SR¹⁰; R³ can be selected from hydrogen and —OR¹¹; R⁴ can be selectedfrom hydrogen and an optionally substituted C₁₋₆ alkyl; R⁵ can behydrogen; R⁶ can be selected from hydrogen and —OR¹¹; and R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ can be independently selected from hydrogenand an optionally substituted C₁₋₆ alkyl.

In some embodiments, R¹, R², R³, R⁴, R⁵ and R⁶ can each be hydrogen. Insome embodiments, R¹ can be methyl; R³ can be —OH; and R², R⁴, R⁵ and R⁶can each be hydrogen. In some embodiments, R³ and R⁶ can each be —OH;and R¹, R², R⁴ and R⁵ and R⁶ can each be hydrogen. In some embodiments,R³ can be —OH; and R¹, R², R⁶ can each be hydrogen. In some embodiments,R¹ and R² can each be —SCH₂CH₂OH; and R³, R⁴, R⁵ and R⁶ can each behydrogen. In some embodiments, R¹ and R² can each be —OCH₃; and R³, R⁴,R⁵ and R⁶ can each be hydrogen. In some embodiments, R¹ can be —OCH₃;and R², R³, R⁴, R⁵ and R⁶ can each be hydrogen. In some embodiments, R¹can be methyl; and R², R³, R⁴, R⁵ and R⁶ can each be hydrogen. In someembodiments, R¹ and R² can each be chloro; and R³, R⁴, R⁵ and R⁶ caneach be hydrogen. In some embodiments, R¹ can be —OH; and R², R³, R⁴, R⁵and R⁶ can each be hydrogen. In some embodiments, R¹ can be phytenyl; R²can be methyl; and R³, R⁴, R⁵ and R⁶ can each be hydrogen. In someembodiments, R¹ and R⁴ can each be t-butyl; and R², R³, R⁵ and R⁶ caneach be hydrogen. In some embodiments, R¹ can be —OH; R² can be—CH₂—CH═C(CH₃)₂; and R³, R⁴, R⁵ and R⁶ can each be hydrogen.

In some embodiments, at least one of R¹, R², R³, R⁴, R⁵ and R⁶ cannot behydrogen. In some embodiments, when R¹ is methyl; and R², R⁴, R⁵ and R⁶are each hydrogen; then R³ cannot be —OH. In some embodiments, when R¹,R², R⁴ and R⁵ are each hydrogen; then at least one of R³ and R⁶ cannotbe —OH. In some embodiments, when R¹, R², R⁴, R⁵ and R⁶ are eachhydrogen; then R³ cannot be —OH. In some embodiments, when R³, R⁴, R⁵and R⁶ are each hydrogen; then at least one of R¹ and R² cannot be—SCH₂CH₂OH. In some embodiments, when R³, R⁴, R⁵ and R⁶ are eachhydrogen; then at least one of R¹ and R² cannot be —OCH₃. In someembodiments, when R², R³, R⁴, R⁵ and R⁶ are each hydrogen; then R¹cannot be —OCH₃. In some embodiments, when R², R³, R⁴, R⁵ and R⁶ areeach hydrogen; then R¹ cannot be methyl. In some embodiments, when R³,R⁴, R⁵ and R⁶ are each hydrogen; then at least one of R¹ and R² cannotbe chloro. In some embodiments, when R², R³, R⁴, R⁵ and R⁶ are eachhydrogen; then R¹ cannot be —OH. In some embodiments, when R² is methyl;and R³, R⁴, R⁵ and R⁶ are each hydrogen; then R¹ cannot be phytenyl. Insome embodiments, when R², R³, R⁵ and R⁶ are each hydrogen; then atleast one of R¹ and R⁴ cannot be t-butyl. In some embodiments, when R²is —CH₂—CH═C(CH₃)₂; and R³, R⁴, R⁵ and R⁶ are each hydrogen; then R¹cannot be —OH.

Examples of compounds of Formula (I) include, but are not limited to thefollowing:

In some embodiments, the compound of Formula (I) can be a dimer, suchthat one of R², R³, R⁴, R⁵ or R⁶ has the structure of Formula (I). Forexample, in some embodiments, the compound of Formula (I) can be Lawsonedimer:

The section below describes some of the conventional therapies that canbe used to inhibit or delay prostate cancer cell growth and/or treat orprevent prostate cancer. It should be understood that the inventivetherapies described herein can be performed with and without any of theconventional therapies for prostate cancer including anyone or more ofthe therapies described in the following section.

III. Prostate Cancer

There were an estimated 192,280 new cases of prostate cancer diagnosedin the U.S. in 2009 and an estimated 27,360 deaths. About 90% ofpatients with advanced disease will develop bone metastases, associatedwith severe pain, loss of mobility, and spinal cord compression. Otheraffected organs may include the liver, lungs and brain. Advancedprostate cancer is resistant to hormone therapy, radiation andconventional chemotherapy. Although the 5-year survival rate is close to100% for local disease, it drops to 30% for advanced cancer.

There have been some advances in the treatment of prostate cancerrecently, including new surgical approaches and improvements inradiotherapy. For example:

1) In 1986, surgeons developed a technique (using da VinciProstatectomy) that allowed the removal of the prostate while minimizingnerve damage, thereby decreasing adverse side effects.

2) In addition, clinical researchers improved a long-establishedradiotherapy technique known as brachytherapy, which involves theimplantation of a small amount of radioactive material (seeds) into theprostate. This radiation therapy method is an effective treatment forearly-stage prostate cancer.

3) There have also been advances in hormonal therapy for prostate cancerincluding the development of gonadotropin-releasing hormone (GnRH)agonists, which inhibit the ability of the pituitary gland to stimulatethe testes to make testosterone.

4) Advances have also been made in chemotherapy for prostate cancer. In2004, results from two large NCI-sponsored clinical trials showed thatuse of the drug docetaxel could prolong the survival of men who hadadvanced prostate cancer which no longer responded to hormonal therapy.

Unfortunately, should the prostate-specific antigen (PSA) level remainabove zero after radical prostatectomy is performed, with conventionaltherapy or with advanced therapy using da Vinci Prostatectomy, thisindicates that the prostate cancer has spread outside the capsule, i.e.,disseminated disease, and to date, there is no curable treatment forthis.

Thus, all current hormonal, as well as, chemotherapy treatment regimensfor such disseminated androgen dependent prostate cancers arepalliative. Subsequently, even if there have been advances in thetreatment of prostate cancer, finding new strategies for treatment ofdisseminated disease remains a crucial challenge. The section belowprovides more details on the use of compounds of Formula (I) to inhibitor delay the growth of cancer cells, in particular prostate cancercells.

IV. Compounds of Formula (I) as Anticancer Agents

Compounds of Formula (I) have significant anti-cancer properties. Forexample, plumbagin (5-hydroxy-2-methylnaphthalene-1,4-dione) is anaturally occurring naphthoquinone that can be found in variousmedicinal herbal species, including Plumbago zeylanica, Staticelimonium, and Limonium carolinianum. Plumbagin has demonstratedanticancer effect toward fibrosarcomas (ED₅₀ 0.75 mg/kg body weight) andP388 lymphocytic leukemia (ED₅₀ 4 mg/kg body weight), induced regressionof hepatoma, and has inhibited growth and invasion of hormone-refractoryprostate cancer. Aziz et al., Cancer Res. 2008, 68(21):9024-322.Furthermore, plumbagin has shown to be a promisingchemopreventive/anticarcinogenic agent against intestinal neoplasia.

Without wishing to be bound by theory, it is contemplated that theprimary mechanism of cytotoxic action of plumbagin and other quinoidcompounds is due to redox-cycling and electrophilic arylation. Plumbagincan be reduced by electron transfer from flavoprotein to a semiquinoneradical, which can, in turn, reduce oxygen to superoxide. The resultingsuperoxide can consequently be converted into hydrogen peroxide,hydroxyl radicals, and/or peroxynitrite, all of which are highlyreactive oxygen species (ROS) with potent cytotoxic and tumoricidialeffects.

While still not wishing to be bound by theory, an additional antitumormechanism of plumbagin and related quinones can involve direct arylationof intracellular thiols leading to depletion of glutathione (GSH).Depletion of GSH may ultimately result in alkylation of cellularmacromolecules and in their inactivation. Moreover, it has been shownthat low dose concentrations of plumbagin (5 umol/L) can inhibitexpression of multiple molecular targets, including protein kinase Cq(PKCq), phosphatidylinositol 3-kinase (PI3K), AKT, activation oftranscription factors activator protein-1 (AP-1), nuclear factor-κB(NF-κB), and signal transducer and activator of transcription 3 (Stat3)in prostate carcinoma cells. Such activities may contribute to thetumoricidial effects of plumbagin.

Studies using plumbagin in pre-clinical models have revealed thattreatment with plumbagin can result in slower growth of androgenindependent prostate cancer, and that the mechanism behind the slowergrowth may be due to apoptosis of prostate tumor cells.

It is contemplated that several compounds of Formula (I) haveanti-cancer activity and that this anti-cancer activity, especially withrespect to prostate cancer, can be significantly improved (e.g., synergycan be obtained) when the compounds are provided in conjunction with ablockade of testosterone/androgen/DHT (e.g., castration, a hormonetreatment therapy, such as hormonal ablation). For example, it isbelieved that the administration of menadione (vitamin K3) to a subjectin need thereof will effectively inhibit the growth of prostate cancercells and thereby reduce the incidence of fatal prostate cancer. Thecombination of menadione with an antioxidant, such as ascorbic acid,alpha lipoic acid, n-acetyl cysteine (NAC), lycopene, tocopherol,tocotrienol, or others may also be beneficial. The combination ofmenadione and mitomycin C can also be beneficial in treating subjectswith advanced solid tumors, advanced lung cancer, and advancedgastrointestinal cancer. By administering a combination of menadione andan antioxidant or plurality of antioxidants, such as vitamin C, tosubjects having prostate cancer, it is contemplated that a reduction intumor cell numbers and PSA (prostate cancer specific antigen) will beobtained.

In a phase I/IIa trial, a combination of menadione and vitamin C weregiven to patients with prostate cancer that had previously failed thestandard of care treatment regimen (i.e., radical prostatectomy,radiotherapy and/or hormonal ablation). Ten of the patients in the trialhad received hormonal ablation therapy prior to the trial but thesepatients were not exposed to hormonal ablation therapy at the time ofreceiving the combination of vitamin C and menadione. See Tareen et al.,Int. J. Med. Sci, 2008, 5:62. In this study, treatment was tested inpatients with late stage disease (aggressive, recurrent). It is likelythat the patients that had previously received hormone therapy hadbecome hormone-resistant at the time of the trial (which is probably whydisease was progressing in these patients).

It is contemplated herein that a significantly improved inhibition ofprostate cancer cell growth can be obtained when castration, hormonalcastration, hormonal ablation, or hormone therapy are provided duringthe time a patient receives the combination of antioxidant (e.g.,ascorbic add) with a compound of Formula (I), such as, menadione.Provided herein is an improved method for treating a subject sufferingfrom prostate cancer with a compound of Formula (I) and androgenablation therapy to subjects with PSA values above zero after radicalprostatectomy, i.e., when they have androgen-dependent disseminateddisease. Today there is no cure for this and patients currently receiveonly palliative treatment, including hormone therapy alone. The dataprovided herein demonstrates that the combination of plumbagin at thetime of hormone therapy is better than hormone-therapy alone.

2,3-Bis[(2-hydroxyethyl)thio]-1,4-naphthoquinone (NSC 95397) can be apotent inhibitor of the dual-specificity phosphatase Cdc25, which isinvolved in cell cycle regulation. NSC 95397 can inhibit the activity ofmitogen-activated protein kinase phosphatases MKP-1 and MKP-3. Thiscompound has been studied in combination with chemotherapy drugs such asdoxorubicin, etoposide, oxaliplatin, and docetaxel. NSC 95397 has beenstudied in neuroendocrine tumor cells, human pancreatic carcinoma cells,and bronchial carcinoma cells. Furthermore, this compound has been usedin prostate cancer cells so as to examine the role of the Cdc25phosphatase in regulation of the mitogen activated protein kinase(MAP-kinase) pathway. See Nemoto et al., Prostate, 2004, 58:95.Nevertheless, the effect of NSC 95397 on the growth or survival ofprostate cancer cells was not reported by Nemoto. It is contemplatedthat NSC 95397 can be used to inhibit prostate cancer cell growth andthat a significantly improved inhibition of prostate cancer cell growthcan be obtained when castration, hormonal castration, hormonal ablation,or hormone therapy are provided before, during, and/or after the time apatient receives the NSC 95397.

Juglone is believed to be a peptidyl-prolyl cis/trans isomerase (PIN-I)inhibitor. Juglone has been studied in combination with etoposide inhuman cancer cells and beta-lapachone can improve the effect ofradiation in laryngeal epidermoid carcinoma cells. It is contemplatedthat the compounds of Formula (I) are highly oxidative and induceoxidative stress in cells. Accordingly, it is contemplated that juglonecan be used to inhibit prostate cancer cell growth and that asignificantly improved inhibition of prostate cancer cell growth can beobtained when castration, hormonal castration, hormonal ablation, orhormone therapy are provided before, during, and/or after the time apatient receives the juglone.

Naphthazarin may be a microtubule depolymerzing agent and2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) may inhibit DNA topoisomerase-I.It is contemplated that naphthazarin and/or2,3-dimethoxy-1,4-naphthoquinone (DMNQ) can be used to inhibit prostatecancer cell growth and that a significantly improved inhibition ofprostate cancer cell growth can be obtained when castration, hormonalcastration, hormonal ablation, or hormone therapy are provided before,during, and/or after the time a patient receives naphthazarin and/or2,3-dimethoxy-1,4-naphthoquinone (DMNQ).

As mentioned above, although treating a subject that has cancer (e.g.,prostate cancer) with one or more compounds of Formula (I) alone or in acombination of compounds of Formula (I) can inhibit the growth ofcancerous cells, a significantly improved inhibition of cancer cellgrowth (e.g., prostate cancer cell growth) can be obtained by providingone or more of the compounds of Formula (I), separately or in a mixtureor combination, in conjunction with a therapy that reduces the androgenlevels of the patient (e.g., castration, hormonal castration, hormonalablation, or hormone therapy). That is, some embodiments include methodsof inhibiting cancer cell growth (e.g., prostate cancer cell growth) ortreating or preventing a cancer (e.g., prostate cancer), wherein asubject having a cancer (e.g., prostate cancer) is provided one or morecompounds of Formula (I) (e.g., plumbagin) while reducing the amount ofandrogens in the subject (e.g., providing castration, hormonalcastration, hormonal ablation, or hormone therapy). Optionally, theinhibition of cancer (e.g., prostate cancer) or a marker thereof (e.g.,PSA) is evaluated after the treatment (e.g., after the combination ofplumbagin and hormone therapy is provided). Stated differently, someembodiments of the invention include a combination of one or more of thecompounds of Formula (1), formulated for administration separately ortogether, and an androgen deprivation therapy (e.g., castration,hormonal castration, hormonal ablation, or hormone therapy) for use ininhibiting or delaying the growth of prostate cancer cells or treatingor preventing prostate cancer. The section below describes some of theapproaches that can be used to deplete the levels of androgen in thesubject so as to provide the treatments and treatment protocolsdescribed above.

V. Hormone Therapy

Hormone therapy for treating prostate cancer, or inhibiting or delayingprostate cancer cell growth, can also be called androgen deprivationtherapy (ADT), chemical castration, or androgen ablation therapy.Androgens can fuel the growth of prostatic cells, including both healthyprostatic cells and cancerous prostatic cells. In some embodiments, asubject suffering from prostate cancer is provided with a hormonetherapy agent that reduces the subject's androgen levels. In someembodiments, the androgen that is decreased in the subject istestosterone, dihydrotestosterone (DHT), androsterone, androstenediol,androstenedione, dehydroepiandrosterone (DHEA), anddehydroepiandrosterone sulfate (DHEA-S). In some embodiments, asubject's serum testosterone level is decreased with one or moreanti-androgen agents or androgen ablation agents. Preferably, theandrogen deprivation therapy is provided during a period in which one ormore compounds of Formula (1) are provided.

In some embodiments, a subject suffering from prostate cancer isclassified as a subject in need of a therapy for prostate cancer andsaid subject is provided a hormone therapy agent that reduces thesubject's androgen levels while said subject is receiving one or morecompounds of Formula (1), such as plumbagin, or a compound presented inTable 1. Optionally, the inhibition in prostate cancer cell growth or aninhibition in prostate cancer advancement is evaluated. Optionally, thedelaying prostate cancer cell growth or delaying prostate canceradvancement is evaluated. A subject can be identified as one in need ofa therapy for prostate cancer using conventional clinical pathologyincluding, biopsy, CT scan, MR!, digital examination, Gleason score, orPSA level. Patients today also get PET scans, which are very importantsince they evaluate the activity of the tumor cells (glucosemetabolism). Similarly, the inhibition or delay of cancer cell growth insaid subject after receiving the treatment can be evaluated usingconventional clinical pathology including, biopsy, CT scan, MRI, digitalexamination, Gleason score, or PSA level.

In some embodiments, the hormone therapy agent that can be used withanyone or more of the methods or treatments described herein is selectedfrom the group consisting of an antiandrogen (including steroidal antiandrogens and nonsteroidal antiandrogens), an estrogen, a luteinizinghormone-releasing hormone (LHRH) agonist, and a LHRH antagonist.Steroidal anti androgen agents include, but are not limited to,cyproterone acetate and finasteride. Nonsteroidal antiandrogens include,but are not limited to, flutamide, nilutamide and bicalutamide. Estrogenagents include, but are not limited to, ethylstilbestrol (DES),megestrol acetate, fosfestrol, and estamustine phosphate. LHRH agonistagents include, but are not limited to, leuprolide, triptorelin,goserelin, histrelin and buserelin. LHRH antagonist agents include, butare not limited to, abarelix and degarelix. Desirably, one or more ofthe compounds selected from the group consisting of cyproterone acetate,finasteride, flutamide, abiraterone, nilutamide, bicalutamide,ethylstilbestrol (DES), megestrol acetate, fosfestrol, estamustinephosphate, leuprolide, triptorelin, goserelin, histrelin, buserelin,abarelix and degarelix are used in the methods and treatments(compositions) described herein, wherein one or more of the compounds ofFormula (I) (e.g., a compound of Table 1) are provided before, during,and/or after providing said cyproterone acetate, finasteride, flutamide,abiraterone, nilutamide, bicalutamide, ethyl stilbestrol (DES),megestrol acetate, fosfestrol, estamustine phosphate, leuprolide,triptorelin, goserelin, histrelin, buserelin, abarelix or degarelix.

As mentioned above, prostate cancer can be treated by hormone therapyagents, however, hormone therapy agents alone can result in thedevelopment of castration-resistant prostate cancer (CRPC). For example,hormonal therapy can initially deliver a response in a subject sufferingfrom prostate cancer, however, the return of hormone-refractory tumorsinvariably prevents long-term patient survival. More effectivestrategies are needed to extend life expectancy and improve the qualityof life for patients with advanced prostate cancer. Accordingly, someaspects of the present invention concern methods for ameliorating orinhibiting or reducing or delaying the onset of castration-resistantprostate cancer (CRPC) or treatments (e.g., compositions used for thepurpose of ameliorating or inhibiting or reducing or delaying the onsetof CRPC), whereby one or more of the compounds of Formula (I) (e.g., acompound from Table 1) are provided before, during and/or afterproviding cyproterone acetate, finasteride, abiraterone, flutamide,nilutamide, bicalutamide, ethyl stilbestrol (DES), megestrol acetate,fosfestrol, estamustine phosphate, leuprolide, triptorelin, goserelin,histrelin, buserelin, abarelix or degarelix. Optionally, the inhibitionin prostate cancer cell growth, an inhibition in prostate canceradvancement, or delaying the onset of CRPC is evaluated. Optionally, apatient with prostate cancer is classified as a subject in need of anagent that ameliorates, reduces, delays, or inhibits the onset of CRPCprior to receiving one or more of the combination therapies describedherein. A subject can be identified as one in need of a therapy forprostate cancer using conventional clinical pathology including, biopsy,CT scan, MRI, digital examination, Gleason score, or PSA level.

Patients today also get PET scans, which are very important since theyevaluate the activity of the tumor cells (glucose metabolism).

Similarly, the inhibition or delay of cancer cell growth in said subjectafter receiving the treatment can be evaluated using conventionalclinical pathology including, biopsy, CT scan, MRI, digital examination,Gleason score, or PSA level. The section below describes the combinationtherapies in greater detail.

VI. Combination Therapies

In some embodiments, the compounds disclosed herein, such as a compoundof Formula (I) (e.g., a compound of Table 1), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition that includes acompound described herein, can be used in combination with one or moreadditional agent(s). Some embodiments disclosed herein relate to amethod of ameliorating or treating a neoplastic disease that can includeadministering to a subject suffering from a neoplastic disease atherapeutically effective amount of one or more compounds describedherein (e.g., a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof), in combination with one or more hormonetherapy agents (referred to as “combination therapy”). Examples ofadditional agents that can be used in combination with a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition that includes a compound of Formula (I), or apharmaceutically acceptable salt thereof, include, but are not limitedto, agents that can decrease the subject's serum androgen levels (e.g.,cyproterone acetate, abiraterone, finasteride, flutamide, nilutamide,bicalutamide, ethyl stilbestrol (DES), megestrol acetate, fosfestrol,estamustine phosphate, leuprolide, triptorelin, goserelin, histrelin,buserelin, abarelix or degarelix).

In some embodiments, the neoplastic disease can be cancer. In someembodiments, the neoplastic disease can be a tumor such as a solidtumor. In an embodiment, the neoplastic disease can be prostate cancerand in some embodiments the prostate cancer can be CRPC. In someembodiments, the prostate cancer is androgen dependent. Therefore, insome embodiments, a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition that includes acompound of Formula (1), or a pharmaceutically acceptable salt thereof,is used in combination with one or more hormone therapy agents for thepurpose of treating a subject that has prostate cancer, for inhibitingthe growth of prostate cancer cells, for delaying prostate cancer, fordecreasing the size of a prostate tumor, or for inhibiting the onset ordevelopment of CRPC.

In some embodiments, a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition that includes acompound of Formula (I) (e.g., one or more of the compounds of Table 1),or a pharmaceutically acceptable salt thereof, is used in combinationwith surgical orchiectomy and/or one or more of the hormone therapyagents cyproterone acetate, finasteride, abiraterone, flutamide,nilutamide, bicalutamide, ethylstilbestrol (DES), megestrol acetate,fosfestrol, estamustine phosphate, leuprolide, triptorelin, goserelin,histrelin, buserelin, abarelix or degarelix such that a “combinationtherapy” is provided.

Normal serum testosterone ranges between 1000-300 ng/dL. In someembodiments, a subject is provided a combination therapy, as describedherein, whereby a reduction in the treated subject's serum testosteronelevel to at least about ≦80, ≦70, ≦60, ≦50, ≦40, ≦30, ≦20, or ≦10 ng/dLis obtained. In some embodiments, a subject is provided a combinationtherapy that reduces the subject's serum testosterone level to at leastabout ≦50 ng/dL. In some embodiments, a subject is treated with acombination therapy that results in a reduction in the subject's serumtestosterone level to at least about ≦20 ng/dL. In some embodiments, asubject is treated with a combination therapy, as described herein, thatreduces the subject's serum testosterone level to at least about or anynumber in between the range of 120-70, 100-60, 80-40, 70-30, 50-20,40-10, 30-10, or 20-10 ng/dL. In some embodiments, a subject is treatedwith a combination therapy that produces a reduction in the subject'sserum testosterone level to about ≦95%, ≦90%, ≦80%, ≦70%, ≦60%, or ≦50%that of a healthy male. In some embodiments, a subject is treated with acombination therapy that results in a reduction in the subject's serumtestosterone level to the range of at least about or any number inbetween the range of about 5-20%, 10-30%, 20-40%, 30-50%, 40-60%, or50-70% that of a healthy male.

Intermittent hormonal therapy (IHT) is an alternative to continuoushormonal therapy, which may delay progression of hormone-refractorydisease (i.e., CRPC). For example, intermittent therapy can be used fora period of 6 months on, followed by a period of 6 months off. In someembodiments, one or more hormonal therapy agents is provided for onemonth on, followed by one month off. In some embodiments, one or morehormonal therapy agents is provided for three months on, followed bythree months off. Accordingly, one or more of the compounds of Formula(I), e.g., a compound of Table 1, can be provided before, during and/orafter IHT, as described above, so as to reduce or inhibit or delay theonset of CRPC.

A non-limiting list of example combination of compounds of Formula (I),or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition that includes a compound described herein, with one or morehormonal therapy agents are provided in Tables 1 and 2. Table 1 providesa shorthand name for each compound of Formula (I) and a shorthand namefor each hormonal therapy agent. Each numbered X compound in Table 2 hasa corresponding compound structure provided in Table 1. Likewise, eachnumbered Y therapy in Table 2 has a corresponding therapy provided inTable 1. Therefore, each “X:Y” entry in Table 2 provides an example of acombination of a compound of Formula (I) and a hormonal therapy agentthat can be used to treat a subject suffering from prostate cancer. Forexample, the combination designated as “F02:AT04” in Table 2 provides acombination of

(plumbagin), and flutamide that can be used to treat a subject sufferingfrom prostate cancer. Each of the combinations provided in Table 2 canbe used with one, two, three or more additional agents described herein.

TABLE 1 Compound of Formula (I) Additional Therapy

cyproterone (AT01) acetate

finasteride (AT02)

bicalutamide (AT03)

flutamide (AT04)

nilutamide (AT05)

bicalutamide (AT06)

ethylstilbestrol (AT07) (DES)

megestrol (AT08) acetate

fosfestrol (AT09)

estamustine (AT10) phosphate

leuprolide (AT11)

triptorelin (AT12)

goserelin (AT13)

histrelin (AT14) — buserelin (AT15) — abarelix (AT16) — degarelix (AT17)— surgical (AT18) orchiectomy

TABLE 2 X:Y X:Y X:Y X:Y X:Y X:Y X:Y F01:AT02 F02:AT02 F03:AT02 F04:AT02F05:AT02 F06:AT02 F07:AT02 F01:AT03 F02:AT03 F03:AT03 F04:AT03 F05:AT03F06:AT03 F07:AT03 F01:AT04 F02:AT04 F03:AT04 F04:AT04 F05:AT04 F06:AT04F07:AT04 F01:AT05 F02:AT05 F03:AT05 F04:AT05 F05:AT05 F06:AT05 F07:AT05F01:AT06 F02:AT06 F03:AT06 F04:AT06 F05:AT06 F06:AT06 F07:AT06 F01:AT07F02:AT07 F03:AT07 F04:AT07 F05:AT07 F06:AT07 F07:AT07 F01:AT08 F02:AT08F03:AT08 F04:AT08 F05:AT08 F06:AT08 F07:AT08 F01:AT09 F02:AT09 F03:AT09F04:AT09 F05:AT09 F06:AT09 F07:AT09 F01:AT10 F02:AT10 F03:AT10 F04:AT10F05:AT10 F06:AT10 F07:AT10 F01:AT11 F02:AT11 F03:AT11 F04:AT11 F05:AT11F06:AT11 F07:AT11 F01:AT12 F02:AT12 F03:AT12 F04:AT12 F05:AT12 F06:AT12F07:AT12 F01:AT13 F02:AT13 F03:AT13 F04:AT13 F05:AT13 F06:AT13 F07:AT13F01:AT14 F02:AT14 F03:AT14 F04:AT14 F05:AT14 F06:AT14 F07:AT14 F01:AT15F02:AT15 F03:AT15 F04:AT15 F05:AT15 F06:AT15 F07:AT15 F01:AT16 F02:AT16F03:AT16 F04:AT16 F05:AT16 F06:AT16 F07:AT16 F01:AT17 F02:AT17 F03:AT17F04:AT17 F05:AT17 F06:AT17 F07:AT17 F01:AT18 F02:AT18 F03:AT18 F04:AT18F05:AT18 F06:AT18 F07:AT18 F08:AT02 F09:AT02 F10:AT02 F11:AT02 F12:AT02F13:AT02 F14:AT02 F08:AT03 F09:AT03 F10:AT03 F11:AT03 F12:AT03 F13:AT03F14:AT03 F08:AT04 F09:AT04 F10:AT04 F11:AT04 F12:AT04 F13:AT04 F14:AT04F08:AT05 F09:AT05 F10:AT05 F11:AT05 F12:AT05 F13:AT05 F14:AT05 F08:AT06F09:AT06 F10:AT06 F11:AT06 F12:AT06 F13:AT06 F14:AT06 F08:AT07 F09:AT07F10:AT07 F11:AT07 F12:AT07 F13:AT07 F14:AT07 F08:AT08 F09:AT08 F10:AT08F11:AT08 F12:AT08 F13:AT08 F14:AT08 F08:AT09 F09:AT09 F10:AT09 F11:AT09F12:AT09 F13:AT09 F14:AT09 F08:AT10 F09:AT10 F10:AT10 F11:AT10 F12:AT10F13:AT10 F14:AT10 F08:AT11 F09:AT11 F10:AT11 F11:AT11 F12:AT11 F13:AT11F14:AT11 F08:AT12 F09:AT12 F10:AT12 F11:AT12 F12:AT12 F13:AT12 F14:AT12F08:AT13 F09:AT13 F10:AT13 F11:AT13 F12:AT13 F13:AT13 F14:AT13 F08:AT14F09:AT14 F10:AT14 F11:AT14 F12:AT14 F13:AT14 F14:AT14 F08:AT15 F09:AT15F10:AT15 F11:AT15 F12:AT15 F13:AT15 F14:AT15 F08:AT16 F09:AT16 F10:AT16F11:AT16 F12:AT16 F13:AT16 F14:AT16 F08:AT17 F09:AT17 F10:AT17 F11:AT17F12:AT17 F13:AT17 F14:AT17 F08:AT18 F09:AT18 F10:AT18 F11:AT18 F12:AT18F13:AT18 F14:AT18

The order of administration of a compound of Formula (I), or apharmaceutically acceptable salt thereof, with one or more additionalhormone therapy agent(s) can vary. In some embodiments, a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, can beadministered prior to all additional hormone therapy agents. In otherembodiments, a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, can be administered prior to at least one additionalhormone therapy agent. In still other embodiments, a compound of Formula(I), or a pharmaceutically acceptable salt thereof, can be administeredconcomitantly with one or more additional hormone therapy agents. In yetstill other embodiments, a compound of Formula (I), or apharmaceutically acceptable salt thereof, can be administered subsequentto the administration of at least one additional hormone therapy agent.In some embodiments, a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, can be administered subsequent to theadministration of all additional hormone therapy agents.

In some embodiments, a subject suffering from prostate cancer is treatedby surgical orchiectomy (i.e., removal of the testes). In someembodiments, a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, can be administered after surgical orchiectomy. In someembodiments, a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, can be administered before and after surgical orchiectomy.

Determining and Evaluating Anti-Cancer Activity

Animal Models

Animal models are pivotal to further our understanding of the mechanismsof (progressive) growth of cancer. Currently used rodent tumor models,including transgenic tumor models, (using genetically modified micesusceptible to develop cancer), as well as implantation of human tumorsunder the skin in immunodeficient mice, do not sufficiently representclinical cancer, especially with regard to metastasis and drugsensitivity. Preclinical tumor model systems employed to evaluatepotential new treatment strategies should aim to represent the processand patterns of metastasis of their clinical counterparts as closely aspossible.

A syngeneic pseudo-orthotopic in vivo model was developed to study theearly steps of prostate cancer. Chambers are surgically placed into thedorsal skinfold of male mice. Briefly, male mice (25-30 g body weight)are anesthetized and placed on a heating pad. Two symmetrical titaniumframes are implanted into the dorsal skinfold. A circular layer isexcised from one of the skin layers. The underlying muscle andsubcutaneous tissues are covered with a glass coverslip incorporated inone of the frames. After a recovery period of 2-3 days, stroma tissueand tumor cells are carefully placed in the chamber.

Tumor-derived cell lines can be grown directly in the chamber,corresponding to the traditional subcutaneous model. However, it wasfound that various minced tissues implanted in the chambers survive andrevascularize, and that tumor-derived cell lines adapt to these variousstroma after co-implantation, which points to this approach as anorthotopic model as well as a model for initial steps in metastasis.

For example, mouse prostate tissue can be grafted in the chamber. Thegraft develops its own vasculature and serve as orthotopic stroma forthe tumor. A small number of prostate cancer cells (e.g., TRAMP-C2 cellsderived from a TRAMP mouse) can be implanted on top of the prostatestroma. The tumor microenvironment can be important for the progressionof different types of cancer, and orthotopic implantation of cancercells can recapitulate human disease much more closely than subcutaneousimplantation. Tumors can grow faster and develop better vasculature whenthe cancer cells are implanted into the relevant organ. Co-implantingmouse prostate cancer cells with prostate stroma can provide the tumorcells with an environment that better reflects the clinical diseasecompared to purely subcutaneous models. Re-vascularized stromal tissueand implanted tumors can remain viable for long periods of time usingthis method, for example, up to 90 days.

Phosphate and Tensin Homolog (PTEN) Deficient Model

Mouse cells derived from the PTEN (phosphatase and tensin homologdeleted in chromosome 10) deficient model of prostate cancer can be usedto study prostate cancer. The tumor suppressor PTEN is one of the mostfrequently mutated genes in human prostate cancer. Loss of PTEN canresult in constitutively high PI3-kinase and Akt activities, which maylead to increased migration, invasiveness, cell proliferation andsurvival. Loss of PTEN can play a major role in the pathogenesis ofhuman prostate cancer. Alteration of at least one PTEN allele isobserved in approximately 60% of primary tumors. Loss of PTEN can beassociated with higher Gleason scores and poor prognosis, cancerprogression toward hormone-independence, resistance to chemotherapy orto radiotherapy, and bone metastasis. PTEN-deficient mice have anincreased incidence of cancer, similarly to the human geneticpredisposition to cancer known as Cowden syndrome, which is caused bygermline mutation in the PTEN gene. In these respects, thePTEN-deficient model appears to mimic human development quite closely.Thus, heterozygous disruption of the PTEN gene can result in spontaneousdevelopment of tumors in several tissues and prostatic intraepithelialneoplasia (PIN) lesions in the prostate. Prostate-specific homozygousloss of PTEN can be sufficient to induce prostate tumors, which canprogress into metastatic disease. Heterozygous loss of PTEN, on theother hand, can cause PIN with a late latency.

Germline homozygous deletion of PTEN may result in embryonic lethalitydue to PTEN ablation. This can be overcome through the conditionalinactivation of the gene using the Cre-LoxP system. A transgenic mousecan be generated that displays expression of the Cre recombinasespecifically in the epithelial cells of the prostate through the use ofthe prostate-specific probasin promoter (PB-Cre4 mice). By crossingthese animals with mice that have floxed PTEN alleles, it can bepossible to generate both heterozygous and homozygous mice in which PTENis deleted specifically in the prostate epithelium. Progression ofprostate cancer in this model is very similar to the progression ofprostate cancer as observed in humans. For example, in this modelepithelial hyperplasia was observed, followed by dysplasia, PIN,invasive adenocarcinoma, and finally metastases to the lymph nodes andto the lung. Similar to human cancer, the PTEN-null mice first regressfollowing androgen ablation, and then become androgen-independent.

Epithelial cell lines can be derived from a prostate tumor dissectedfrom a homozygous PTEN^(L/L)/PBCre+ mouse. At least two clonal celllines (PTEN-P2 and PTEN-P8) are heterozygous PTEN^(L/+). The remainingallele can be silenced by forced expression of the Cre recombinase invitro (PTEN-CaP2 and PTEN-CaP8 cells). Loss of the second allele canincrease anchorage-independent growth and confer tumorigenesis in vivo.Spontaneous androgen-independence can occur in vivo, even though thePTEN-CaP2 and PTEN-CaP8 cells express the androgen receptor.

The implementation of PTEN prostate cells in the animal models disclosedherein can be highly relevant to human prostate cancer, and can allowdetailed observation of the growth and/or regression of prostate tumorsin response to different treatment regimens. Implantation in syngeneicmice respects many aspects of normal tumor growth. For example, twopairs of mouse prostate cancer cells (PTEN-P2/8 and PTEN-CaP2/8) canfacilitate examination of metastasis in a mouse model of prostate cancerthat is relevant to human cancer

IntraVital Microscopy (IVM)

IntraVital Microscopy (IVM) can be used to visualize tumors in animalsand analyze various aspects of cancer physiology such as tumorvascularization, cell migration and metastasis. An advantage of IVMincludes the real-time analysis of dynamic processes with single-cellresolution. IntraVital microscopy offers the possibility to follow tumorgrowth in a non-invasive, non-destructive manner. The application of IVMcan be limited to animal models that bear visually accessible tumors.Therefore, the dorsal skinfold chamber model described above can becompatible with IVM. Using IVM can permit a number of parameters to bemeasured in living animals and as a function of time, including tumorgrowth, angiogenesis, infiltration by immune cells, tumor cellmigration, cell cycle entry, mitosis (cell-division) and apoptosis(programmed cell death), all in the context of the host and in realtime.

VIII. Pharmaceutical Compositions

Some embodiments described herein relate to a pharmaceuticalcomposition, that can include a therapeutically effective amount of aone or more compounds described herein (e.g., a compound of Formula (I),(e.g., a compound in Table 1) or a pharmaceutically acceptable saltthereof, and/or a hormone therapy agent) and a pharmaceuticallyacceptable carrier, diluent, excipient or combination thereof. In someembodiments, the pharmaceutical composition can include a singlediastereomer of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, (for example, a single diastereomer is presentin the pharmaceutical composition at a concentration of greater than 99%compared to the total concentration of the other diastereomers). Inother embodiments, the pharmaceutical composition can include a mixtureof diastereomers of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof. For example, the pharmaceutical composition caninclude a concentration of one diastereomer of >about 50%, ≧60%, ≧70%,≧80%, ≧90%, ≧95%, or ≧98%, as compared to the total concentration of theother diastereomers. In some embodiments, the pharmaceutical compositionincludes a racemic mixture of diastereomers of a compound of Formula(I), or a pharmaceutically acceptable salt thereof.

Some embodiments described herein relates to a pharmaceuticalcomposition, that can include a therapeutically effective amount acompound of Formula (I), an additional hormone therapy agent, and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof. Some embodiments described herein relates to a pharmaceuticalcomposition, that can include a therapeutically effective amount acompound of Formula (1), and a pharmaceutically acceptable carrier,diluent, excipient or combination thereof. Some embodiments relate to apharmaceutical composition that can include a therapeutically effectiveamount of a hormone therapy agent and a pharmaceutically acceptablecarrier, diluent, excipient or combination thereof.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orcarriers, diluents, excipients or combinations thereof. Properformulation is dependent upon the route of administration chosen.Techniques for formulation and administration of the compounds describedherein are known to those skilled in the art.

The pharmaceutical compositions disclosed herein may be manufactured ina manner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes. Additionally, theactive ingredients are contained in an amount effective to achieve itsintended purpose. Many of the compounds used in the pharmaceuticalcombinations disclosed herein may be provided as salts withpharmaceutically compatible counterions.

Multiple techniques of administering a compound and/or agent exist inthe art including, but not limited to, oral, rectal, topical, aerosol,injection and parenteral delivery, including intramuscular,subcutaneous, intravenous, intramedullary injections, intrathecal,direct intraventricular, intraperitoneal, intranasal and intraocularinjections.

One may also administer the compound and/or agent in a local rather thansystemic manner, for example, via injection of the compound directlyinto the infected area, often in a depot or sustained releaseformulation. Furthermore, one may administer the compound and/or agentin a targeted drug delivery system, for example, in a liposome coatedwith a tissue-specific antibody. The liposomes will be targeted to andtaken up selectively by the organ.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions that can include a compound and/oragent described herein formulated in a compatible pharmaceutical carriermay also be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition.

IX. Dosing

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight, the severity of theaffliction, and mammalian species treated, the particular compoundsemployed, and the specific use for which these compounds are employed.The determination of effective dosage levels, that is the dosage levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods, for example, human clinicaltrials and in vitro studies.

The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Alternatively dosages may be based andcalculated upon the surface area of the patient, as understood by thoseof skill in the art. Although the exact dosage will be determined on adrug-by-drug basis, in most cases, some generalizations regarding thedosage can be made. The daily dosage regimen for an adult human patientmay be, for example, an oral dose of between 0.01 mg and 3000 mg of eachactive ingredient, preferably between 1 mg and 700 mg, e.g. 5 to 200 mg.The dosage may be a single one or a series of two or more given in thecourse of one or more days, as is needed by the subject. In someembodiments, an active ingredient will be administered for a period ofcontinuous therapy, for example for a week or more, or for months oryears. In some embodiments, an active ingredient can be administered onetime per day.

Multiple doses can be administered to a subject. For example, an activeingredient can be administered once per month, twice per month, threetimes per month, every other week (qow), once per week (qw), twice perweek (biw), three times per week (tiw), four times per week, five timesper week, six times per week, every other day (qod), daily (qd), twice aday (qid), or three times a day (tid), over a period of time rangingfrom about one day to about one week, from about two weeks to about fourweeks, from about one month to about two months, from about two monthsto about four months, from about four months to about six months, fromabout six months to about eight months, from about eight months to about1 year, from about 1 year to about 2 years, or from about 2 years toabout 4 years, or more.

In some embodiments, a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof, and a hormone therapy agent can be cyclicallyadministered to a patient. Cycling therapy involves the administrationof a first active ingredient for a period of time, followed by theadministration of a second active ingredient for a period of time andrepeating this sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more therapies, avoid or reduce theside effects of one or more therapies, and/or improve the efficacy oftreatment. In some embodiments, a compound of Formula (I) or apharmaceutically acceptable salt thereof, and a hormone therapy agentare administered in a cycle of less than about 3 weeks, about once everytwo weeks, about once every 10 days, or about once every week. Thenumber of cycles can be from about 1 to about 12 cycles, or from about 2to about 10 cycles, or from about 2 to about 8 cycles.

In some embodiments, the active ingredient can be a compound of Formula(I), or a pharmaceutically acceptable salt thereof. In some embodiments,the active ingredient can be a hormone therapy agent. In someembodiments, both an active ingredient of compound of Formula (I), or apharmaceutically acceptable salt thereof, and an active ingredient of ahormone therapy agent are administered to a subject.

The daily dosage regimen for an adult human patient may be the same ordifferent for two active ingredients provided in combination. Forexample, a compound of Formula (I) can be provided at a dose of between0.01 mg and 3000 mg, while a hormone therapy agent can be provided at adose of between 1 mg and 700 mg. The dosage or each active ingredientcan be, independently, a single one or a series of two or more given inthe course of one or more days, as is needed by the subject. In someembodiments, the active ingredients will be administered for a period ofcontinuous therapy, for example for a week or more, or for months oryears. In some embodiments, a compound of Formula (I), or apharmaceutically acceptable salt thereof, can be administered one timeper day. In some embodiments, the hormone therapy agent can beadministered once a week.

In instances where human dosages for active ingredients have beenestablished for at least some condition, those same dosages may be used,or dosages that are between about 0.1% and 500%, more preferably betweenabout 25% and 250% of the established human dosage. Where no humandosage is established, as will be the case for newly-discoveredpharmaceutical compositions, a suitable human dosage can be inferredfrom ED₅₀ or ID₅₀ values, or other appropriate values derived from invitro or in vivo studies, as qualified by toxicity studies and efficacystudies in animals.

In cases of administration of a pharmaceutically acceptable salt,dosages may be calculated as the free base. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the active ingredients disclosed herein in amounts thatexceed, or even far exceed, the above-stated, preferred dosage range inorder to effectively and aggressively treat particularly aggressivediseases or infections.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each active ingredient but can be estimated from in vitrodata. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations. Dosageintervals can also be determined using MEC value. Compositions should beadministered using a regimen which maintains plasma levels above the MECfor 10-90% of the time, preferably between 30-90% and most preferablybetween 50-90%. In cases of local administration or selective uptake,the effective local concentration of the drug may not be related toplasma concentration.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Active ingredients disclosed herein can be evaluated for efficacy andtoxicity using known methods. For example, the toxicology of aparticular active ingredient, or of a subset of the active ingredients,sharing certain chemical moieties, may be established by determining invitro toxicity towards a cell line, such as a mammalian, and preferablyhuman, cell line. The results of such studies are often predictive oftoxicity in animals, such as mammals, or more specifically, humans.Alternatively, the toxicity of particular compounds in an animal model,such as mice, rats, rabbits, or monkeys, may be determined using knownmethods. The efficacy of a particular active ingredient may beestablished using several recognized methods, such as in vitro methods,animal models, or human clinical trials. When selecting a model todetermine efficacy, the skilled artisan can be guided by the state ofthe art to choose an appropriate model, dose, route of administrationand/or regime.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1

Compounds of Formula (I) can be prepared by methods known in the art.Additionally, many compounds of Formula (I) are naturally occurringorganic compounds that can be isolated from plants. Furthermore, manycompounds of Formula (I) are commercially available.

Plumbagin is soluble in alcohol, acetone, chloroform, benzene, andacetic acid. Plumbagin has been used in preparation with Ethanol (invitro) and in preparation with DMSO (in vitro) or DMSO with PEG 30% (invivo).

Example 2 Cell Culture

PTEN-P2/GFP are cells that stably express histone H2B-GFP fusionprotein. Kanda et al. (Kanda T, Sullivan K F, Wahl G M. Histone-GFPfusion protein enables sensitive analysis of chromosome dynamics inliving mammalian cells. Curr Biol 1998 Mar. 26; 8(7):377-85) developed ahighly sensitive method for observing chromosome dynamics in livingcells. They fused the human Histone H2B gene to the gene encoding theGFP, which was transfected into human HeLa cells to generate a stableline constitutively expressing H2B-GFP. The H2B-GFP fusion protein wasincorporated into chromatin without affecting cell cycle progression. Wehave generated cDNA encoding a Histone H2B-GFP fusion protein under the5′LTR in the LXRN retroviral cassette, and have introduced it into anumber of humans, as well as, murine cancer cell lines by retroviraltransduction.

Cells are grown in DMEM medium containing 10% FBS, 2 mM L-glutamine, 100U/ml penicillin/100 ng/ml streptomycin, insulin-selenium-transferrin (5ng/ml insulin), and DHT 10⁻⁸M final. Androgen withdrawal is achieved bykeeping the cells in phenol red-free DMEM medium containing 10%charcoal-treated FBS and the same supplements as in the normal mediumexcept for DHT. Cells are maintained in a humidified incubator at 37° C.and 5% CO₂. G418 (100 ng/ml) is added to maintain stable expression ofH2B-GFP.

Cell Counting:

Cells in 12-well plates are washed once with PBS, detached usingTrypsin, and transferred to a suspension vial in a final volume of 10 mlPBS. Cells are counted using a COULTER™ Multisizer II instrument(Beckman Coulter Inc., Hialeah, Fla.) gated for the appropriate cellsize and corrected for particulate debris.

Animal Model and Surgical Techniques:

Animal experiments have been approved as appropriate. All surgicalprocedures are performed in a sterile laminar flow hood. Dorsal skinfoldchambers and surgical instruments are autoclaved before use. Saline usedto keep tissue moist during surgical preparation is mixed withgentamicin (50 μl/ml).

Male Nude mice (25-35 g body weight) are anesthetized (7.3 mg ketaminehydrochloride and 2.3 mg xylazine/100 g body weight, i.p.) and placed ona heating pad. Two symmetrical titanium frames are implanted into adorsal skinfold, so as to sandwich the extended double layer of skin. A15 mm full thickness circular layer is excised. The underlying muscle(M. cutaneous max.) and subcutaneous tissues are covered with a glasscoverslip incorporated in one of the frames. After a recovery period of2-3 days, prostate tissue and cancer cell spheroids are carefully placedin the chamber. Small circular Band Aids are applied on the backside ofthe chamber after surgery to prevent scratching. Before surgery,Buprenorphine (0.1 mg/kg) will be given IP. After surgery Meloxicam willbe given in the drinking water for 4 days Meloxicam (5.0 mg/ml), isadded at 35 μl per 100 ml of water to be medicated.

Preparation of Stroma:

A male donor mouse is euthanized and the anterior prostate tissue isexcised, put in a Petri dish with antibiotics (gentamicin 50 μl/ml), andminced with fine scissors into small pieces (<1 mm²) for implantation.

Preparation of Tumor Spheroids:

Liquid overlay plates are generated using 1% Agarose melted in DMEM thatis added to round-bottom 96-well plates (50 ul/well). Cancer cells grownas pre-confluent monolayers are trypsinized, diluted to a final volumeof 250,000 tumor cells/ml. Viability is determined using Trypan blue.The cells are plated at 100 ul/well into the agarose-coated plates.After 48 hrs the cells form spheroids, which are picked and washed inserum-free medium before implantation into the mouse chambers. Viabilityis determined using Trypan blue. The size of the implanted spheroid canbe determined precisely to minimize variations between animals.

Surgical Castration:

Mice are anesthetized with 7.3 mg ketamine hydrochloride and 2.3 mgxylazine/100 g body weight, i.p. A lateral incision across the scrotumis made and the testes are individually ligated and excised. The woundwas cauterized. The incision was then sutured and sealed with Nexaband®acrylic.

Intravital Microscopy:

Fluorescence microscopy is performed using a Mikron InstrumentMicroscope equipped with epi-illuminator and video-triggeredstroboscopic illumination from a xenon arc (MV-7600, EG&G). A siliconintensified target camera (SIT68, Dage-MTI) is attached to themicroscope. A Hamamatsu image processor (Argus 20) with firmware version2.50 (Hamamatsu Photonic System) is used for image enhancement and forthe capture of images to a computer. A Zeiss Plan Neoflour 1.25X/0.035objective is used to obtain an over-view of the chamber and to determinetumor size. A Zeiss A-Plan 10X/0.25 objective is used to capture imagesfor calculation of vascular parameters. A Zeiss Achroplan 20X/0.5 Wobjective is used to capture images for calculation of mitotic andapoptotic indices. Our system permits evaluation of the followingparameters.

Tumor Area

(A_(T)) is defined as number of pixels with photo density above 75 (256gray levels), i.e., A_(T)=ΣA_(k), for 75<k<255.

Number of Tumor Cells:

When tumors are heterogeneous, changes in A_(T) do not directly reflecttumor growth. An estimate of the number of tumor cells (N_(TC)) can beobtained by fitting to a quadratic function of an intensity index, e.g.N_(TC)=−3.296×10⁻¹²+190.6×I_(T)+7.7310⁻²×(I_(T))², where the index ofintensity is given by I_(T)=ΣA_(k)*k for 75<k<255.

Mitotic and Apoptotic Indices:

At each time point, two peripheral and two central ×20 fields of thetumor are captured with a FITC filter and an integrated frame grabber.Only mitotic figures in metaphase-telophase (MI) are included in themitotic indices to exclude the potential artifact of nuclear membranedistortion. Apoptotic/Pyknotic nuclei are defined as H₂B-GFP labelednuclei with a cross sectional area <30 μm². Nuclear karyorrhexis (NK),easily distinguishable by the vesicular nuclear condensation andbrightness of H2BGFP, is included within this apoptotic indices.

Image Analysis of Vascular Parameters:

For each spheroid, video recordings are used to calculate length, areaand vascular density of the neovasculature being induced by theimplanted tumor spheroids. Vascular parameters are analyzed from thevideo recording using Image-Pro Plus. Photomicrographs obtained with the×10 objective, are “flattened” to reduce the intensity variations in thebackground pixels. An Area of Interest (AOI) is selected to eliminatedistorted areas, and thresholding is used to segment the picture intoobjects and background. This panel is used to calculate the vasculararea (A_(v)). The picture is skeletonized to calculate the vascularlength (L_(v)). The average tumor vessel diameter D_(v) is calculated asA_(v)/L_(v), and the vascular density (A_(v)) is calculated as L_(v) pertumor area. Finally, we calculate the growth rate of the total area oftumor vasculature.

Example 3 Effect of Naphthoquinone Analogs on PTEN-P2/GFP CellProliferation

PTEN-P2/GFP prostate cancer cells were plated at a density of 8000cells/well in 96-well plates (triplicates) in growing medium containing10% Fetal Bovine Serum and DHT. The next day, increasing concentrationsof a naphthoquinone analog (diluted from 10 mM DMSO stock solutions)were incubated for 24 hrs. Cell viability was determined by theformazan-based cytotoxicity assay “CellTiter96Aquaeous nonradioactiveproliferation assay” (Promega). The results are shown in Tables 3 and 4,and FIGS. 1 and 2.

TABLE 3 2-methoxy- 1,4- 1,4- Phyllo- naphtho- naphtho- NSC quinonequinone quinone 95397 (K1) conc % % % % (μM) viability σ viability σviability σ viability σ 0 100.0 3.6 100.0 2.2 100.0 3.7 100.0 2.7 1100.0 3.3 93.1 3.9 103.7 5.6 107.9 6.1 2 97.0 0.7 88.4 1.3 100.6 4.4108.1 5.2 3 93.9 2.6 86.9 1.9 86.6 5.1 106.4 7.1 4 90.4 1.8 85.0 3.268.4 6.3 106.8 8.1 5 90.1 0.9 82.5 1.0 53.8 3.5 108.0 6.8 7 88.7 1.367.2 2.2 47.1 4.3 110.8 7.5 10  83.0 2.5 43.5 8.2 36.3 1.7 110.1 9.2 25 47.3 3.0 0.3 0.4 15.1 7.5 109.1 7.4 50  0.1 0.5 −0.4 0.2 4.8 1.0 109.88.2 2,6- di-tert- butyl- 1,4-naphto- Lawsone quinone Lapachol dimerJuglone conc % % % % (μM) viability σ viability σ viability σ viabilityσ 0 100.0 1.7 100.0 3.2 100.0 2.1 100.0 3.0 1 103.9 3.0 106.4 6.2 98.32.4 90.7 2.7 2 104.5 4.7 105.3 6.1 98.6 2.3 85.1 3.1 3 104.7 6.3 102.47.0 97.4 3.6 80.6 3.6 4 103.0 7.2 102.8 5.6 95.1 2.9 80.5 1.2 5 104.06.3 99.8 4.8 96.8 3.1 58.2 6.0 7 102.8 2.7 99.7 6.5 96.1 3.7 2.3 2.3 10 102.2 6.2 99.6 5.4 98.6 1.7 1.1 0.5 25  102.9 3.6 96.2 5.6 99.4 1.8 2.50.2 50  92.9 6.0 86.1 4.4 97.9 1.8 5.3 1.9 Naphtha- Mena- zarin dioneDMNQ Lawsone conc % % % % (μM) viability σ viability σ viability σviability σ 0 100.0 3.6 100.0 2.5 100.0 3.3 100.0 3.4 1 73.4 5.6 95.03.1 95.0 3.9 90.1 1.3 2 36.4 2.6 93.1 1.8 91.0 1.5 85.1 1.9 3 8.9 4.690.2 3.7 88.5 2.1 81.3 3.5 4 1.5 0.7 93.5 1.7 85.9 3.4 82.5 3.5 5 1.70.6 89.4 3.7 85.3 7.9 80.4 3.5 7 2.2 0.6 94.0 2.0 58.3 4.2 80.3 2.9 10 2.4 0.9 77.5 7.0 2.4 1.8 83.1 1.9 25  5.4 0.7 2.4 0.7 0.9 0.6 87.0 2.450  9.1 0.5 2.9 0.7 0.8 0.5 96.0 1.5 Plum- Dichlon bagin conc % % (μM)viability σ viability σ 0 100.0 3.3 100.0 0.8 1 96.5 2.0 94.6 2.2 2 95.61.7 90.9 3.3 3 92.3 3.6 88.9 2.1 4 92.2 2.3 73.0 0.7 5 91.8 1.7 32.9 6.07 99.3 4.2 0.4 0.4 10  87.1 1.1 0.6 0.2 25  89.2 3.0 — — 50  4.9 2.3 — —

TABLE 4 Compound IC50 (μM) Naphtazarin 1.65 Plumbagin 4.55 Juglone 5.3NSC 95397 6.2 DMNQ 7.35 2-methoxy-1,4- 8.95 naphtoquinone Menadione 14.51,4-naphtoquinone 24.1 Dichlon 37.75 Phylloquinone (K1) >502,6-di-tert-butyl-1,4- >50 naphtoquinone Lapachol >50 Lawson >50 Lawsonedimer >50

Example 4 Dose Response Plumbagin PTEN-P2/GFP Cells

PTEN-P2/GFP mouse cancer cells were placed in androgen withdrawal mediumin the presence or absence of DHT (dihydrotestosterone) at a finalconcentration of 10⁻⁸ M. Plumbagin was added at the indicatedconcentrations for 24 hours. The absence of DHT simulates surgical orchemical castration. Cells were trypsinized and counted using a CellCoulter counter Multisizer II, which excludes debris. Results representcell numbers as percent of control (in which the number of cells in theabsence of drug is 100%). FIG. 3 is a graph that shows the mean of twoseparate experiments, each run in duplicates. The results are shown inTable 5 and FIG. 3. The results indicate that in vitro, the combinationtreatment of plumbagin with simulated surgical or chemical castrationwas more efficient than treatment with plumbagin alone.

Androgen withdrawal medium: DMEM high-glucose phenol-red free, with thefollowing additives: 10% charcoal-treated Fetal Bovine Serum, 25 ug/mlbovine pituitary extract, 5 ug/ml insulin, 6 ng/ml EGF recombinant.

TABLE 5 μM plumbagin % control % control Average noDHT 0 100.01 100.00100.00 1 73.70 95.96 84.83 2 42.90 37.14 40.02 4 10.12 1.57 5.84 8 0.450.22 0.33 with DHT 0 100.00 100.00 100.00 1 106.93 61.29 84.11 2 94.1819.16 56.67 4 22.62 4.89 13.76 8 0.85 0.40 0.62

Example 5 In Vivo Effect of Plumbagin Combined with Castration in thePseudo-Orthotopic Chamber Model for Prostate Cancer

Platinum chambers were placed in the dorsal skinfold of nude mice bysurgery. Two days later, minced prostate tIssUe from BalbC mice(syngeneic) was grafted into the chambers and allowed to vascularize for7 to 10 days. Small tumor cells spheroids were implanted into eachchamber. Tumor cells PTEN-P2 stably transfected with H2B-GFP fusionprotein (PTEN-P2/GFP) were used in these experiments. When tumorvascularization was established (about 5-7 days), the animals weresurgically castrated to inhibit androgen production. Surgical castrationinduces androgen deprivation, and is known in the art to effectivelymimic clinical hormone therapy. The mice were treated with plumbaginsoon after castration. Plumbagin administration schedule was 1 mg/kg(DMSO and PEG30%) via intraperitoneal injection, once/day. The resultsunexpectedly indicate that the combination treatment of plumbagin withcastration was more efficient in vivo than either treatment alone.Therefore, this experiment provides an important indication thatcastration (whether surgical or chemical) in combination with plumbagincan provide a significant improvement over therapies that werepreviously known in the art.

Furthermore, the results demonstrate that treatment with castrationonly, or treatment with plumbagin only, did not lead to a markeddecrease in tumor size. However, the combination treatment of castrationwith plumbagin unexpectedly resulted in significant decreases in tumorsize. As such, the combination therapies described herein providesignificant improvements in treating prostate cancer over therapies thatwere previously known in the art.

FIG. 4 compares the growth of tumors without treatment, castrationalone, plumbagin alone, and the combination of castration and plumbagin.

FIG. 5 shows the effect of plumbagin at 0.1 mg/kg, 0.3 mg/kg and 1mg/kg, given in combination with castration. In FIGS. 4 and 5, day 0 isthe first day of plumbagin treatment.

Example 6 In Vivo Effect of Plumbagin Combined with Castration in thePseudo-Orthotopic Chamber Model for Prostate Cancer

Platinum chambers were placed in the dorsal skinfold of nude mice bysurgery. Two days later, minced prostate tissue from BalbC mice(syngeneic) was grafted into the chambers and allowed to vascularize for7 to 10 days. Small tumor cells spheroids were implanted into eachchamber. Tumor cells PTEN-P2 stably transfected with H2B-GFP fusionprotein (PTEN-P2/GFP) were used in these experiments. The animals weresurgically castrated about three weeks after implantation to inhibitandrogen production. Surgical castration induces androgen deprivation,and is known in the art to effectively mimic clinical hormone therapy.Two weeks after castration, the mice were treated daily with plumbaginat 2 mg/kg ip.

FIG. 6 illustrates the effect of adding plumbagin after surgicalcastration.

FIG. 7 illustrates increasing apoptosis CAP) and mitosis (MI) afterdaily administration of plumbagin ip (2 mg/kg). This figure illustratesthat underlying the rapid tumor regression, there was an increase; rAapoptosis, but also that mitosis increased, which was interpreted ascell cycle arrest.

The results unexpectedly indicate that the combination treatment ofplumbagin with castration was more efficient in vivo than castrationalone. Therefore, this experiment provides an important indication thatcastration (whether surgical or chemical) in combination with plumbagincan provide a significant improvement over therapies that werepreviously known in the art.

Furthermore, the results demonstrate that treatment with castrationonly, did not lead to a marked decrease in tumor size. However, thecombination treatment of castration with plumbagin unexpectedly resultedin significant decreases in tumor size. As such, the combinationtherapies described herein provide significant improvements in treatingprostate cancer over therapies that were previously known in the art.

Without wishing to be bound by theory, the observations indicate that

tumor regression is likely caused by a combination of decreasedvascularization due to androgen withdrawal, together with tumor cellgrowth arrest or with tumor cells apoptosis due mostly to plumbagintreatment. Thus, the efficacy of the combination was much better in vivothan can be observed in vitro because the separate effects of eachtreatment on distinct biological compartments (vasculature stromapossibly inflammatory cells) are not represented in the culture of celllines.

Example 7 Dose Response Plumbagin in Human LNCaP Cells

LNCaP hormone-sensitive human prostate cancer cells were placed inandrogen withdrawal medium in the absence of DHT (dihydrotestosterone).The absence of DHT simulates surgical or chemical castration. Theandrogen withdrawal medium was phenol-red free DMEM high-glucosecontaining 10% charcoal-treated Fetal Bovine Serum.

Plumbagin was added at the indicated concentrations in Table 6 for 24hours. Cells were trypsinized and counted using a Cell Coulter counterMultisizer II, which excludes debris. The results in Table 6 representcell numbers as percent of control (in which the number of cells in theabsence of drug is 100%). FIG. 8 illustrates the effect of plumbagin inhuman LNCaP cells. The results indicate that in vitro, the combinationtreatment of plumbagin with simulated surgical or chemical castration ismore efficient than treatment castration alone.

TABLE 6 Plumbagin cell number/20 cone. (μM) trial 1 trial 2 trial 3trial 4 trial 5 trial 6 trial 7 mean % control 0 7245 7376 7551 76037327 8047 7562 7530 100 0.5 6422 5989 6453 6475 — — — 6335 84.13 1 69977139 6769 6490 — — — 6849 90.95 2 5324 5282 4522 4821 — — — 4987 66.23 43005 3082 3327 3300 — — — 3179 42.21 6 1396 1500 1323 1352 — — — 139318.50 8 330 283 284 287 — — — 296 3.93

Example 8 In Vivo Effect of Plumbagin Combined with Chemical Castration

Platinum chambers are placed in the dorsal skinfold of nude mice bysurgery. Two days later, minced prostate tissue from BalbC mice(syngeneic) are then grafted into the chambers and allowed tovascularize for 7 to 10 days. Small tumor cells spheroids are thenimplanted into each chamber. Tumor cells PTEN-P2 stably transfected withH2B-GFP fusion protein (PTEN-P2/GFP) are then used in these experiments.When tumor vascularization is established (about 5-7 days), the animalsare treated with an antiandrogen compound (e.g., cyproterone acetate) toinduce androgen deprivation. The mice are then treated with plumbagin oran analog thereof (e.g., a compound from Table 1). Plumbagin or analogthereof is administered according to the schedule: 1 mg/kg (DMSO andPEG30%) via intraperitoneal injection, once/day. Control mice that arenot treated with cyproterone acetate are analyzed in parallel. Also,mice treated with cyproterone acetate, but not treated with plumbaginare analyzed in parallel. The results will show that the combination ofplumbagin (or analog thereof) with the anti androgen compound (e.g.,cyproterone acetate) will inhibit prostate cancer cell growth moreefficiently than treatment with plumbagin (or analog thereof) orantiandrogen compound (e.g., cyproterone acetate) alone.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present disclosure. Therefore, it should be clearly understood thatthe forms disclosed herein are illustrative only and are not intended tolimit the scope of the present disclosure.

What is claimed is:
 1. (canceled)
 2. A product combination that inhibitsor delays the growth of prostate cancer, wherein the product combinationcomprises: a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt of Formula (I):

wherein: le is methyl; R² is hydrogen; R³ is —OH; R⁴ is hydrogen; R⁵ ishydrogen; R⁶ is; and an androgen deprivation therapy agent that reducesthe production of testosterone.
 3. The product combination of claim 2,wherein the androgen deprivation therapy agent is a luteinizinghormone-releasing hormone (LHRH) agonist or a LHRH antagonist.
 4. Theproduct combination of claim 2, wherein the androgen deprivation therapyagent is selected from the group consisting of leuprolide, triptorelin,goserelin, abarelix, degarelix, and abiraterone.
 5. The productcombination of claim 2, wherein the androgen deprivation therapy agentdecreases the subject's serum testosterone level to about 5-20 of ahealthy male subject.
 6. The product combination of claim 2, whereinsaid product combination inhibits the growth of prostate cancer.
 7. Theproduct combination of claim 2, wherein said product combinationinhibits or delays the onset of castration-resistant prostate cancer. 8.The product combination of claim 2, wherein the androgen deprivationtherapy agent is abiraterone.
 9. The product combination of claim 2,wherein the androgen deprivation therapy agent is leuprolide.
 10. Theproduct combination of claim 2, wherein the androgen deprivation therapyagent is degarelix.
 11. The product combination of claim 2, wherein thecompound of Formula (I) is administered to the subject orally.
 12. Theproduct combination of claim 2, wherein the compound of Formula (I) andthe androgen deprivation therapy agent are provided to a subject in asingle formulation or a single dosage.
 13. The product combination ofclaim 2, wherein the androgen deprivation therapy agent is administeredto the subject orally.
 14. The product combination of claim 2, whereinthe compound of Formula (I) and the androgen deprivation therapy agentare administered to the subject orally.
 15. The product combination ofclaim 2, wherein said prostate cancer is androgen dependent prostatecancer.
 16. The product combination of claim 2, wherein said prostatecancer is castration-resistant prostate cancer.
 17. The productcombination of claim 2, wherein the androgen deprivation therapy agentdecreases the subject's serum testosterone level to at least about ≦50ng/dL.
 18. The product combination of claim 2, wherein the androgendeprivation therapy agent decreases the subject's serum testosteronelevel to at least about ≦20 ng/dL.
 19. The product combination of claim2, wherein the product combination results in a decrease in prostatecancer tumor size.